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    Research Grants Awarded

    Role of GluR6 in cell senescence and in the development of breast cancer

    Study Section:
    Tumor Cell Biology I

    Scientific Abstract:
    Breast cancer cells can proliferate indefinitely, whereas normal diploid breast epithelial cells undergo replicative senescence after a finite number of generations. Cellular senescence is regulated through the functioning of multiple genes and escape from senescence is hypothesized as an essential early step in tumor progression. Recently, molecular pathways leading to cell senescence have been contemplated as potential targets for cancer therapy. Applying a functional approach, we cloned a gene, SEN6A, which can restores normal cell growth leading to senescence in breast tumor cells. Homology search of human genome database identified SEN6A as a previously known gene, GluR6, which encodes for ionotropic kainate glutamate receptor 6. Prior to our discovery, it was thought that GluR6 expression was limited to central nervous system, where it is an important receptor for exitatory neurotransmitters. We found that GluR6 is expressed in most tissues; its expression is elevated in late passage, relative to young human cells; and it is either absent or expressed at low level in breast tumor cells. Our preliminary results show that ectopic expression of GluR6 restores normal cell growth pattern and senescence in breast cancer cells. Our results and chromosomal location of GluR6 at 6q21, a LOH hot spot, indicate that GluR6 is a senescence gene that may be involved in the etiology of breast cancer. Several DNA markers, used in various LOH studies, are located within GluR6 gene. The goal of this project is to establish the role of GluR6 gene in the regulation of normal cell multiplication and development of breast cancer. The specific aims of this proposal are: 1. Analyze effect of ectopic expression of GluR6 on the growth of breast tumor cells. GluR6 gene, cloned in an ecdysone inducible expression retroviral vector system, in frame with GFP, will be introduced into breast tumor cell lines. Gene transfer clones will be analyzed for growth inhibition, stage of cell cycle arrest, telomerase activity and telomere length and for the expression of other growth related proteins by western blot analyses. 2. Analyze breast tumor tissues and cell lines for abnormal expression or mutations in GluR6 gene. We will apply denaturing HPLC and sequencing analysis for mutational screen. 3. Identify molecular pathways associated with GluR6 expression through antibody microarray technology and yeast two-hybrid screen. Defining the role of GluR6 in molecular pathway(s) leading to senescence would identify important targets involved in the onset and progression of breast cancer, thus would provide new markers for early diagnosis and therapeutic intervention. This proposal addresses basic question of molecular mechanisms involved in the onset and progression of breast cancer hence is directly related to objectives of the breast cancer research program.

    Lay Abstract:
    Breast cancer cells can proliferate indefinitely in body or in culture, while normal cells stop growing and become senescent after a finite number of divisions. Since senescence restricts cell multiplication, it is an important defense mechanism against cancer. It is regulated through multiple genes and escape from senescence is an essential early step in tumor progression. Recently, molecular pathways leading to cell senescence have been contemplated as potential targets for cancer therapy. In our ongoing studies, we cloned a human gene, SEN6A, which restores normal cell growth pattern leading to senescence in breast tumor cells. Homology search of human genome database identified SEN6A as a previously known gene, GluR6, which encodes for glutamate receptor 6. Prior to our discovery, it was thought that GluR6 expression was limited to central nervous system, where it is an important receptor for exitatory neurotransmitters. We found that GluR6 is expressed in most tissues; it is expressed at higher lever in senescent cells, relative to young human cells; and gene is turned off in many breast tumor cell lines. Introduction of a cloned GluR6 gene, into breast cancer cells, induces growth arrest leading to senescence. These findings and the location of GluR6 at a region of chromosome 6, which is frequently deleted in breast cancer, suggest that GluR6 is a senescence gene that may be involved in the etiology of breast cancer. The goal of this project is to establish that GluR6, is indeed involved in the regulation of normal cell growth, and alterations in GluR6 gene are related to the development of breast cancer. Specific Aims: 1. A normal copy of GluR6 will be cloned in an inducible vector system and introduced into breast cancer cells in culture. The inducible vector system will allow GluR6 to be turned on or off as required in the experiments. Tumor cells, expressing the transferred gene, will be analyzed for the restoration of normal cell growth pattern and senescence. The same cell population will be examined for growth resumption when the gene is turned off. 2. We will apply microarray technology to identify genes that are turned on or off in response to GluR6 expression and identify molecular pathways associated with GluR6 expression. 3. We will analyze breast tumor tissues and cell lines to catalog mutations in GluR6 that may be responsible for its aberrant expression in breast tumors. In tumor cells where GluR6 is intact but inactive, we will try to find out why and look for ways to reactivate it. This proposal addresses basic question of molecular mechanisms involved in the onset and progression of breast cancer hence is directly related to objectives of the breast cancer research program. Proposed studies will verify GluR6 as a tumor suppressor gene and establish its role in the development of breast tumors. This project will identify new markers for early diagnosis and targets for innovative therapeutic strategies.